Hepatocellular carcinoma (HCC) develops synergistically in HCV-infected patients with alcoholism, and no treatments are available for this devastating complication. Our study reveals the role of TLR4-dependent Nanog+ cancer stem cells (CSCs) in liver oncogenesis induced by alcohol in HCV Ns5aTg mice. Defective TGF-2 signaling in 22 spectrin heterozygote (22sp+/-) mice, leads to aberrant IL-6 and Nanog induction and spontaneous HCC development. These two mechanistic concepts are merged by our recent findings that the Nanog+ CSCs have defective TGF-2 signaling, and 22sp+/- mouse livers have TLR4 induction. Further, CDK4 which is activated by TLR4 signaling, may disrupt TGF-2 signaling via its interaction with 22SP and Smad3, preventing Smad complex nuclear translocation. In fact, CDK4 haploinsufficiency (Cdk4+/-) attenuates HCC incidence in 22sp+/- mice, suggesting the causal role of CDK4 in inactivating TGF-2 pathway and consequently promoting liver oncogenesis. Based on these results, we propose a ground-breaking hypothesis that reciprocal regulation of heightened TLR4 oncogenic signaling and defective TGF-2 tumor suppressor pathway are causally linked via CDK4 to render synergistic liver oncogenesis. To test this hypothesis, we will exploit the use of the putative Nanog+ CSCs and cross-utilization of Ns5aTg, 22sp+/-, and Cdk4+/- mice and pursue the following specific aims: 1) to determine the role of activated CDK4 and defective TGF-2 signaling in TLR4-dependent oncogenic activity of the Nanog+ CSCs isolated from liver tumors of alcohol-fed HCV Ns5aTg mice;2) to test whether alcohol feeding increases HCC incidence in 22sp+/- mice in a manner dependent on TLR4;3) to test whether defective TGF-2 signaling aggravates TLR4-dependent liver oncogenesis in alcohol-fed Ns5aTg:22sp+/- compound mice;and 4) to determine whether CDK4 haploinsufficiency or a novel CDK4 inhibitor protects alcohol-fed Ns5aTg:22sp+/- mice from HCC. This program will meet the charge of the Recovery Act by immediately hiring 3 new full-time laboratory personnel and retaining 2 full-time scientists. Further, results from the proposed basic and pre-clinical studies will identify the interactive causal roles of CDK4, TGF-2 and TLR4 signaling in liver oncogenesis and will lead to the development of anti-CDK4, pro-TGF-2, and anti-TLR4 modalities for prevention and treatment of HCC, which itself holds enormous potential for an economic stimulus. PUBLIC HEALTH RELEVANCE: The proposed collaborative research will test a ground-breaking mechanism of liver cancer development caused by alcohol and hepatitis virus infection with a main emphasis on convergence of two reciprocally affected signaling pathways. This research will identify new therapeutic targets for liver cancer caused by HCV and alcohol, leading to development of novel preventive and therapeutic drugs.